The androgen receptor (AR) is a member of the nuclear receptor superfamily. It is a transcription factor that plays a critical role in normal hormonal signaling as well as in the development of prostate cancer. Nearly all primary prostate cancers express the androgen receptor, hence androgen deprivation is the cornerstone of therapy. When prostate cancer recurs, it does so in an androgen independent form, usually still expressing the AR. The AR is the center of a multi-protein complex, the components of which depend on the presence or absence of ligand. Identifying and characterizing these proteins is essential to the development of strategies of controlling prostate cancer. Using the yeast two-hybrid system, a number of proteins have been shown to interact with the AR, some of which can modulate gene transcription in overexpression systems. However, the entire AR complex has not been examined under physiologic conditions in prostate (or any other) cells. The last five years has seen dramatic advances in mass spectrometry (MS) based protein study. This field has benefited from rapidly enlarging protein databases and advances in computer processors. Using MS, it is now possible to determine the identity of the proteins present in complex biological samples. This proposal will examine the hypothesis that co-regulator proteins associated with the androgen receptor directly determine its transcriptional activity in androgen rich and starved conditions, and that androgen independence results from alteration of this multi-protein complex. The aims of this proposal are to use a novel mass spectrometry based proteomic method to identify the proteins present in endogenous AR complexes immunoprecipitated from prostate cancer cells in androgen rich and androgen starved states. The same technique will be applied to high dose androgen stimulation, a model for androgen induced differentiation, and to an androgen independent cell line, a model for recurrent disease. Identified proteins will be tested for interaction using co-precipitation and mammalian 2-hybrid systems and for modulation of AR mediated transcriptional activity using a reporter system. Androgen dependent and independent prostate cancer specimens will be tested for identified proteins using real-time PCR and immunohistochemistry.